芜菁花青素合成关键酶DFR基因启动子的克隆及功能分析

 在已经克隆的‘津田’芜菁和‘赤丸’芜菁二氢黄酮醇4–还原酶（DFR）基因的基础上，通过染色体步移法从两种芜菁基因组中克隆了BrDFR1和BrDFR2基因上游1 296和1 297 bp的启动子序列。生物信息学分析表明，启动子片段中包含TATA box、CAAT box、光调控元件、ABA应答元件、伤害应答元件、低温应答元件、防御与胁迫应答元件、MeJA应答元件等多个顺式作用元件。启动子BrDFR1P和BrDFR2P仅在15个核苷酸位点存在差异。将BrDFR1P和BrDFR2P分别连接到pCAMBIA1301植物表达载体上，构建Promoter::GUS载体，通过农杆菌介导遗传转化烟草。GUS组织化学染色检测表明，BrDFR1P和BrDFR2P均能驱动GUS基因表达。构建BrDFR1P和BrDFR2P的一系列缺失体，融合GUS基因后遗传转化烟草。染色结果表明，BrDFR1P和BrDFR2P缺失片段均具有相应的起始下游基因转录的活性特征。

Cloning and Functional Analysis of the Key Enzyme DFR Promoters in Turnip Anthocyanin Biosynthesis

Based on the gene sequences of dihydroflavonol 4-reductase（DFR）which were cloned from‘Tsuda’and‘Yurugi Akamaru’turnip，the 1 296 and 1 297 bp promoter fragments of BrDFR1 and BrDFR2 genes were obtained from these two turnips’ genome by genome walking method. More cis-acting elements of BrDFR1 and BrDFR2 promoters，such as TATA box，CAAT box，light responsive elements，ABRE，WUN-motif，LTR，TC-rich repeats and MeJA responsive motif，were identified using bioinformatics method. The nucleotide sequences of BrDFR1P and BrDFR2P had 15 differences. The pCAMBIA1301 plant expression vectors containing these two promoter sequence respectively were constructed with the GUS reporter gene，which were called Promoter::GUS vectors，and then transformed into tobacco through the mediation of Agrobacterium tumefaciens. Histochemical staining analysis showedthat the expression of GUS reporter gene could be driven by BrDFR1P and BrDFR2P. A series of 5′-end deleted fragments of these two promoters were constructed，which were fused with GUS gene and then transformed into tobacco. The stain result indicated that the deleted fragments of BrDFR1P and BrDFR2P had relatively active characteristic that could start the expression of GUS gene.